Methods for modulating transcriptional activation using mint proteins

ABSTRACT

The present invention is directed to isolated nucleic acids encoding Mint protein variants having enhanced abilities to modulate the transcriptional activation mediated by the cytoplasmic tail of the amyloid precursor protein (APP) relative to wild-type Mint proteins. The present invention is further directed toward purified Mint protein variants having enhanced abilities to modulate the transcriptional activation mediated by the cytoplasmic tail of APP relative to wild-type Mint proteins. The present invention also encompasses methods of modulating transcriptional activation and methods of identifying compounds that modulate transcriptional activation, and vectors, as well as transfected cells and kits useful for modulating transcriptional activation or for the identification of compounds that can modulate transcriptional activation. The present invention further encompasses transgenic knockout mice with little or no expression of Mint 1, Mint 2 or Mint 3 proteins. Such reagents may be useful as candidate therapeutics for Alzheimer&#39;s disease (AD), or as models for the rational design of drugs useful for the treatment of AD.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/227,490, filed Aug. 23, 2002, now U.S. Pat. No. 7,081,337 which is acontinuation-in-part of U.S. patent application Ser. No. 09/821,861,filed Mar. 30, 2001 and issued as U.S. Pat. No. 6,649,346, to each ofwhich priority is claimed, and each of which is incorporated byreference in its entirety herein.

The subject matter described herein was supported, at least in part, byfunds provided by the United States Government. Accordingly, the UnitedStates Government may have certain rights to this invention.

INTRODUCTION

The present invention relates to methods for modulating transcriptionalactivation and hence to methods for the treatment of Alzheimer's disease(AD). The invention is based, at least in part, on the observation that,while all three MsX2-interacting nuclear target (Mint) proteins (Mints1-3) can bind to the cytoplasmic tail of APP, only Mints 1 and 2modulate the transcriptional activation mediated by the cytoplasmic tailof APP.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is a degenerative brain disorder that ischaracterized clinically by progressive loss of memory and cognitiveimpairment. Pathologically, the disease is characterized by lesionscomprising neurofibrillary tangles, cerebrovascular amyloid deposits,and neuritic plaques. The cerebrovascular amyloid deposits and neuriticplaques contain amyloid-β peptide. The aggregation of amyloid-β peptidemay be instrumental in the pathogenesis of AD.

Amyloid-β peptide is derived from amyloid-β precursor protein (APP). APPis a ubiquitous type 1 membrane protein (Kang et al., 1987, Nature325:733-736; Kitaguchi et al., 1988, Nature 331:530-532; Tanzi et al.,1988, Nature 331:528-30) that is physiologically processed byproteolytic cleavage. (Selkoe, 1998, Trends Cell Biol 8: 447-453; Bayeret al., 1999, Mol Psychiatry 4:524-528; Haass and De Strooper, 1999,Science 286:916-919; Wolfe and Haass, 2001, J Biol Chem 276:5413-5416).First, cleavage by α- or β-secretases releases the large extracellularportion of APP. Subsequently, the remaining sequences of APP composed ofa small extracellular stub, the transmembrane region (TMR), and thecytoplasmic tail are digested by γ-secretase at multiple positions. SeeSastre et al., 2001, EMBO Rep 2:835-841; Yu et al., 2001, J Biol Chem276:43756-43760. γ-Cleavage liberates an intracellular cytoplasmicfragment that may be translocated to the nucleus (Cupers et al., 2001, JNeurochem 78:1168-1178; Kimberly et al., 2001, J Biol Chem276:40288-40292) and may function as a transcriptional activator (Caoand Südhof, 2001, Science 293:115-120; Gao and Pimplikar, 2001, ProcNatl Acad Sci USA 98:14979-14984). In addition, γ-cleavage generatessmall peptides derived from the TMR and adjacent extracellular sequencesthat include Aβ40 and Aβ42 which form the amyloid fibrils in Alzheimer'sdisease. See Glenner and Wong, 1984, Biochem Biophys Res Commun122:1131-1135; Masters et al., 1985, EMBO J 4:2757-2763; reviewed inSelkoe, 1998, Trends Cell Biol 8: 447-453; Haass and De Strooper, 1999,Science 286:916-919.

The γ-cleavage of APP is mediated by presenilins, intrinsic membraneproteins that may correspond to γ-secretase and that are mutated in somecases of familial AD. See, e.g., Esler et al., 2000, Nat Cell Biol2:428-434. Also, γ-cleavage occurs in APP homologs that are notimplicated in AD. For example, Notch proteins are membrane proteins thatare also cleaved in the middle of the TMR in a presenilin-dependentreaction. See, e.g., Yea et al., 1999, Nature 398:525-529; De Strooperet al., 1999, Nature 398:518-522; Struhl et al., 1999, Nature398:522-525. Notch proteins are cell-surface proteins involved inintercellular signaling in which presenilin-dependent cleavage liberatesa cytoplasmic fragment that functions in nuclear transcription. Struhlet al., 2000, Mol Cell 6:625-636. Sterol regulatory element bindingproteins (SREPPs) are also cleaved to generate nuclear transcriptionfactors. Brown et al., 2000, Cell 100:391-398. These observationssuggested that the short cytoplasmic tail fragment of APP also mayfunction as a transcriptional activator, and that feedback loops mayexist between the nucleus and the cytoplasm or cell membrane whereby therate of APP proteolysis is regulated.

This first of these hypotheses was confirmed by the findings that theshort cytoplasmic tail of APP contains an NPTY (SEQ ID NO: 17) sequencethat binds to phosphotyrosine binding (PTB) domains in multipleproteins, including Fe65 and Mints/X11a. See Fiore et al., 1995, J BiolChem 270:30853-30856; Borg et al., 1996, Mol Cell Biol 16:6229-6241;Guenette et al., 1996, Proc Natl Acad Sci USA 93:10832-10837; McLoughlinand Miller, 1996, FEBS Lett 397:197-200; Zhang et al., 1997, EMBO J. 16:6141-6150. Fe65 is an adaptor protein that forms a transcriptionallyactive complex with the released APP tail and a nuclear histoneacetyltransferase, Tip60. See Cao and Südhof, 2001, Science 293:115-120.

In published United States patent application 20010034884, Perausmodified APP to create an APP fusion protein that incorporated a Gal4binding domain and a VP16 transactivating domain, so that the rate offormation of the cytoplasmic tail of APP could be monitored by measuringthe level of expression of a reporter gene whose transcription wascontrolled by a regulatable promoter containing Gal 4 binding sites.However, in contrast to the study of Cao and Südhof (2001), Perausexpressed no appreciation that the cytoplasmic tail of APP, withoutmodification, acted as a transcription factor under physiologicalconditions, through an interaction with Fe65 and Tip60.

As introduced above, the NPTY sequence in the cytoplasmic tail fragmentof APP can facilitate the binding of this protein to the PTB domainsthat are present in proteins of the Mints/X11 family. Mints 1 and 2 aregenes initially identified as candidates for Friedreich's ataxia. Basedon partial sequence analysis, these genes were thought to be orthologs.See Duclos and Koenig, 1995, Mamm Genome 6: 57-58. However, thesequencing of full-length cDNAs showed that the encoded proteins wereproducts of distinct genes. See Okamoto and Südhof, 1997, J Biol Chem272:31459-31464. To prevent confusion among different types of X11s,these proteins were named Mints 1 and 2. A third isoform was dubbed Mint3. See Okamoto and Südhof, 1997, J Biol Chem 272:31459-31464; Okamotoand Südhof, 1998, Eur J Cell Biol 77:161-165. Subsequent recloning ofthe same proteins led to further renaming, and they are now alsovariably referred to as X11α/β/γ, mLin-10s, X11a/b/c, or X11L1/L2.

Mints/X11 proteins are composed of a long isoform-specific N-terminalsequence, a central PTB domain, and two C-terminal PSD-95, Drosophiladisc large, zona occludens (PDZ) domains. Mint proteins interact withseveral other proteins in addition to APP. Mint 1 (but not Mints 2 or 3)binds to calcium/calmodulin-dependent serine protein kinase (CASK) (Butzet al., 1998, Cell 94:773-782), another adaptor protein (Hata et al.,1996, J Neurosci 16: 2488-2494). In C. elegans, CASK and Mint 1 homologsare encoded by the Lin-2 and Lin-10 genes whose mutation causes similarvulvaless phenotypes, suggesting that the Mint 1/CASK complex isevolutionarily conserved. See Butz et al., 1998, Cell 94:773-782; Kaechet al., 1998, Cell 94:761-771; Borg et al., 1998b, J Biol Chem273:31633-31636; and Borg et al., 1999, J Neurosci 19:1307-1316. Mints 1and 2 also bind to Munc18-1, an essential fusion protein at the synapse(Okamoto and Südhof, 1997, J Biol Chem 272:31459-31464; Biederer andSüdhof, 2000, J Biol Chem. 275:39803-39806; Verhage et al., 2000,Science 287:864-869), and to presenilins which are intrinsic componentsof the γ-secretase (Lau et al., 2000, Mol Cell Neurosci 16:557-565).

The functions of the Mint proteins remain obscure. In C. elegans, Lin-10(Mint 1) mediates the correct targeting of EGF-like receptors to thebasolateral membrane of vulval precursor cells (Whitfield et al., 1999,Mol Biol Cell 10:2087-2100), and is necessary for delivery of AMPA-likeglutamate receptors to synapses (Rongo et al., 1998, Cell 94:751-759).These data suggest that Lin-10/Mint 1 functions in membrane traffic ofproteins to specific plasma membrane domains. In vertebrates, however, avariety of somewhat contradictory functions for Mints have beenproposed. Transfection experiments revealed that Mints alter productionof Aβ peptides, indicating a role in APP cleavage. See Borg et al.,1998a, J Biol Chem 273:14761-14766; Sastre et al., 1998, J Biol Chem273:22351-22357; Mueller et al., 2000, J Biol Chem 275:39302-39306. Incontrast, an interaction of Mint 1 with KIF17 in vitro led to theproposal that Mint 1 functions in trafficking neuronal NMDA-, but notAMPA-type glutamate receptors in vertebrates. See Setou et al., 2000,Science 288:1796-1802. This study renamed Mint 1 “mLin-10” in analogy tothe C. elegans gene, but did not reference the previous finding that innematodes Lin-10 only affects AMPA- but not NMDA-receptors (Rongo etal., 1998, Cell 94:751-759).

The small size of the APP cytodomain and the overlapping of its regionsinvolved in the binding of Fe65 and the Mints proteins suggest that thelatter may be involved in the competitive regulation of theintracellular signaling events mediated by the cytoplasmic tail of APP.Because it is likely that the rate of APP proteolysis is directly orindirectly regulated through feedback mechanisms that operate betweenthe nucleus and the cytoplasm or cell membrane, a better understandingof the interactions between the cytoplasmic tail of APP and the Mintsproteins will expand our knowledge of the mechanisms whereby the rate ofAPP proteolysis is regulated, thereby leading to greater insight intothe pathophysiology of AD.

These studies may also facilitate the development of new methods oftreatment for this disease. At present, the only medications approved bythe U.S. Food and Drug Administration for the treatment of AD arecholinesterase inhibitors. Unfortunately, these drugs provide onlylimited clinical benefit in controlling the symptoms of AD, and dolittle to actually intervene in the disease process. Thus, there is astrong need for the development of better treatments for AD.

In accordance with the present invention, it has been discovered that,while all three Mint proteins (Mints 1-3) bind to the cytoplasmic tailof APP, only Mint 1 and 2 can modulate the transcriptional activationmediated by a fusion protein consisting of the cytoplasmic tail of APPcoupled to the transcription factor Gal4/VP16. These findings suggestthat Mints 1 and 2, variants of these proteins that display enhancedabilities to modulate the transcriptional activation mediated by thecytoplasmic tail of APP relative to wild-type Mints 1 and 2, or smallmolecules which either mimic, inhibit or potentiate the effects of theseproteins in modulating the transcriptional activation mediated by thecytoplasmic tail of APP, may be useful in regulating the rate of APPproteolysis and hence in the treatment of AD.

SUMMARY OF THE INVENTION

The present invention is directed to isolated nucleic acids encodingMint protein variants having enhanced abilities to modulate thetranscriptional activation mediated by the cytoplasmic tail of theamyloid precursor protein (APP) relative to wild-type Mint proteins. Inpreferred embodiments, the nucleic acids are those having the nucleotidesequences of SEQ ID NOS:1-8.

The present invention is further directed to purified Mint proteinvariants having enhanced abilities to modulate the transcriptionalactivation mediated by the cytoplasmic tail of APP relative to wild-typeMint proteins. In preferred embodiments, these proteins are those havingthe amino acid sequences of SEQ ID NOS:9-16.

The present invention further provides a method of modulatingtranscriptional activation. In one embodiment, this method comprisesintroducing into a target cell a nucleic acid encoding a wild-type Mintprotein or a Mint protein variant having enhanced abilities to modulatetranscriptional activation relative to wild-type Mint proteins. Inpreferred embodiments, the nucleic acids are those having the nucleotidesequences of SEQ ID NOS:1-8. In another embodiment, this methodcomprises introducing into a target cell a wild-type Mint protein or aMint protein variant having enhanced abilities to modulatetranscriptional activation relative to wild-type Mint proteins. Inpreferred embodiments, these proteins are those having the amino acidsequences of SEQ ID NOS:9-16. The Mint proteins and Mint proteinvariants of the present invention modulate transcriptional activationmediated by the cytoplasmic tail of APP.

The present invention also provides a method of identifying compoundsthat modulate transcriptional activation comprising contacting a cellcontaining an APP molecule modified in the C-terminal cytoplasmic tailto permit the specific transcriptional activation of a reporter gene andmeasuring the levels of reporter gene transcription in the presence andabsence of the compound, wherein increased or decreased levels ofreporter gene transcription in the presence of the compound indicatethat the compound is capable of modulating transcriptional activation.Such compounds may be useful as candidate therapeutics for AD, or asmodels for the rational design of drugs useful for the treatment of AD.

The present invention further provides for transgenic knockout mice forMint 1, Mint 2 and Mint 3. These animals may be useful for elucidatingthe pathophysiology of AD and for developing improved treatments forthis disease.

In other embodiments, the present invention is directed to vectors,transfected cells and kits useful for modulating transcriptionalactivation or for the identification of compounds that can modulateAPP-mediated transactivation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-B shows the similarly tight binding of Mints 1, 2, and 3 to thecytoplasmic tail of APP. A. Immunoblot analysis of Mint binding to theimmobilized cytoplasmic tail of APP. B. Quantitation of the amount ofMints bound to the immobilized cytoplasmic tail of APP as percent ofMints in the starting brain extract.

FIG. 2A-B show that co-transfection of Mints 1, 2, or 3 increases thesteady-state levels of APP. A. Immunoblot analysis of HEK293 cellsco-transfected with expression plasmids encoding APP₆₉₅, Mints 1-3, andFe65 as indicated. B. Quantitation of the levels of APP₆₉₅ intransfected HEK293 cells shown in A.

FIG. 3A-C show the binding of both PDZ domains of Mints to thecytoplasmic C-terminal sequence of presenilins. A. Binding of Mints 1and 3 to presenilin 1. B. Percent of Mints solubilized from a ratforebrain membrane preparation that bound to an affinity columncontaining a peptide corresponding to the C-terminus of presenilin 1 ora control peptide derived from gp41 (n=3). C. Binding of various Mint 1mutants prepared from transfected COS cells to the immobilizedcytoplasmic C-terminal sequence of presenilin 1. Mint 1 mutantscontaining inactivating point mutations in either the first (Mint 1PDZ1*), the second (Mint 1 PDZ2*), or both PDZ domains (Mint 1 PDZ1/2*),or a truncation mutant of Mint 1 lacking the two PDZ domains (Mint 1ΔPDZ) were analyzed by affinity chromatography on immobilized presenilin1 or gp41 control peptides.

FIG. 4A-F shows the immunoreactivity of Mint 1, Mint 2 and APP in mousehippocampus. Vibratome sections (35 μm) from adult mouse hippocampuswere stained for Mint 1 (A and D), Mint 2 (B and E), and APP (C and F)using the avidin-biotin peroxidase method.

FIG. 5A-L shows the immunofluorescence localization of Mints 1 and 2 incultured hippocampal neurons. The left (red) and center (green) picturesshow the separate fluorescence channels from double immunofluorescencelabeling experiments, whereas the right pictures show the merged images.Neurons were labeled with the following antibodies: A-C, antibodies toMint 1 (A) and Mint 2 (B). Note that the pictures show alow-magnification overview with a high-magnification inset. D-F,antibodies to synapsins (D) and Mint 2 (E). G-I, antibodies to APP (G)and Mint 1 (H). J-L, antibodies to APP (J) and Mint 2 (K). Scale bar inpanel (applies to the low-magnification views in A-C)=50 μm; scale barin panel 1 (applies to the insets in A-C, and the full images in allother pictures)=20 μm.

FIG. 6A-L shows that Mints 1 and 2 are concentrated in the trans-Golgicomplex. Subcellular distribution of Mints has been further investigatedwith a group of well-characterized markers to Golgi apparatus,endoplasmic reticulum and early endosome. Cultured hippocampal neuronswere double-labeled with antibodies to the following proteins: A-C, Mint2 (A), TGN38 (B), and merged images (C); D-F, Mint 1 (D), the Golgi 58Kprotein (E), and merged images (F); G-I, Mint 2 (G), calnexin (H), andmerged images (I); J-L, Mint 2 (J), EEA1 (K), and merged images (L).Scale bar in L (applies to all images)=15 μm.

FIG. 7A-B shows the use of APP-Gal4/VP16 fusion proteins to measureγ-cleavage of APP. A. Relative luciferase activity in PC12 cellsfollowing transfection with plasmids containing the constructs indicatedin B.

FIG. 8A-B shows the sequence requirements of APP γ-cleavage measured byGal4/VP16-dependent transactivation. A. Requirements of extra- andintracellular APP sequences for transactivation. B. Test of the specificfunctions of extra- vs. intracellular APP sequences in γ-cleavage, andof the effect of a dominant-negative mutant of presenilin 2 (PS2DA;Steiner et al., 1999, J Biol Chem 274:28669-28673).

FIG. 9A-C shows that Mints 1 and 2 but not Mint 3 inhibittransactivation by APP-Gal4/VP16 fusion proteins. A. Dose-dependentinhibition by Mint 1 of wild-type APP-Gal4/VP16 transactivation but notof APP-Gal4/VP16 carrying a point mutation in the cytoplasmic NPTYbinding sequence for Mints (APP*-GV). B. Effects of Mints 1, 2, and 3 onAPP-Gal4/VP16 dependent transactivation. C. Quantitated immunoblotanalysis of the Mints expressed in the experiment shown in B.

FIG. 10A-C shows the structure-function analysis of Mint 1 indicatingthe role of PDZ domains in inhibiting APP-Gal4/VP16 dependenttransactivation. A. Transactivation as a function of proteinconcentration. B. Transactivation observed in HEK293 cellsco-transfected with APP-Gal4/VP16 and a control plasmid, or wild-typeMint 1, a Mint 1 mutant lacking the PDZ-domains, wild-type Mint 3, or aMint 3 mutant lacking both PDZ domains. C. Immunoblot analysis of theMint mutants analyzed in B to control expression of the constructs inthe experiments shown.

FIG. 11A-C show that the PTB and PDZ domains of Mint 1 cooperate ininhibiting transactivation by APP-Gal4/VP16. A. Effect ofco-transfecting Mint 1 and various Mint 1 point-mutants in thePTB-domain and the first PDZ domain with APP-Gal4/VP16. B. Effect ofdeleting the N-terminal isoform-specific Mint 1 sequences on inhibitionof transactivation. C. Quantitated immunoblot analysis of Mintexpression in the experiment shown in B.

FIG. 12 shows the binding-dependent effect of Mints on transactivationmediated by a fusion protein of the intracellular fragment of APP withGal4/VP16.

FIG. 13A-P shows the sequences of the various Mint 1 and 2 nucleic acidsand peptides. A. Mint 1 cDNA (SEQ ID NO:1). B. Mint 1 PDZ1* cDNASequence (SEQ ID NO:2). C. Mint 1 ΔPDZ cDNA Sequence (SEQ ID NO:3). D.Mint 1 Δ N-term cDNA Sequence (SEQ ID NO:4). E. Mint 2 cDNA Sequence(SEQ ID NO:5). F. Mint 2 PDZ1* cDNA Sequence (SEQ ID NO:6). G. Mint 2 ΔPDZ cDNA Sequence (SEQ ID NO:7). H. Mint 2 Δ N-term cDNA Sequence (SEQID NO:8). I. MINT 1 Peptide Sequence (SEQ ID NO:9). J. MINT 1 PDZ1*Peptide Sequence (SEQ ID NO:10). K. MINT 1 Δ PDZ Peptide Sequence (SEQID NO:11). L. MINT 1 Δ N-term Peptide Sequence (SEQ ID NO:12). M. MINT 2Peptide Sequence (SEQ ID NO:13). N. MINT 2 PDZ1 *Peptide Sequence (SEQID NO:14). O. MINT 2 Δ PDZ Peptide Sequence (SEQ ID NO:15). P. MINT 2 ΔN-term Peptide Sequence (SEQ ID NO:16).

FIG. 14A-C depicts the strategies used for the production of transgenicmice in which the function of the Mint 1 (A), Mint 2 (B) or Mint 3 (C)gene has been eliminated. In each case, schematic representations of thewild-type gene, the targeting vector and the mutant gene are provided.

FIG. 15A-B shows Southern blot analysis of Mint 1 knockouts. A. Genomictail DNA from offspring of heterozygous interbreedings was digested withSpeI and hybridized with the outside probe shown in FIG. 14A. The upperband corresponds to wild-type allele and the lower to the mutant allele.B. Southern blot analysis of Mint 1 knockouts. Genomic tail DNA fromoffspring of heterozygous interbreedings was digested with SpeI andhybridized with a probe to the first exon shown in FIG. 14A.

DETAILED DESCRIPTION OF THE INVENTION

Proteolytic processing of the amyloid precursor protein (APP) produces,inter alia, amyloid-β peptide, which may contribute to the pathogenesisof Alzheimer's disease (AD), and a C-terminal cytoplasmic tail, whichmay act as a transcriptional activator. In accordance with the presentinvention, it has been discovered that the proteins Mint 1, Mint 2 andMint 3 can strongly bind to the PDZ domain of the C-terminal cytoplasmictail of APP (see Example #1). Moreover, it has been discovered that thebinding of Mint 1 and Mint 2 to the cytoplasmic tail of APP stronglyinhibits the transcriptional activation produced by this protein (seeExample #6). In particular, it has been discovered that thetranscriptional activation of a reporter gene whose transcription isregulated by the binding of the GAL4/VP-16 transcription factor by anAPP-Gal4/VP16 fusion protein can be strongly inhibited by Mint 1 andMint 2 (see Example #6). This observation suggests that Mint proteins,especially Mint 1 and 2, variants of these proteins with enhancedabilities to modulate transcriptional activation relative to wild-typeMint proteins, or other molecules that may mimic the effects of theMints or inhibit or potentiate the interaction between Mint proteins andthe cytoplasmic tail of APP may be useful either as candidatetherapeutics for the treatment of AD, as reagents for the identificationof candidate therapeutics for the treatment of AD, or as models ortargets for rational drug design.

The present invention relates to isolated nucleic acids encoding Mintproteins or Mint protein variants having enhanced abilities to modulatetranscriptional activation relative to wild-type Mint proteins. Inpreferred embodiments, the nucleic acids are those having the nucleotidesequences of SEQ ID NOS:1-8. In other preferred embodiments, the nucleicacids are nucleic acids encoding the polypeptides having the amino acidsequences of SEQ ID NOS:9-16. In other embodiments, the nucleic acidsare nucleic acids comprising the nucleic acids having the nucleotidesequences of SEQ ID NOS:1-8 or the nucleic acids encoding thepolypeptides having the amino acid sequences of SEQ ID NOS:9-16. Instill further embodiments, the nucleic acids are those substantiallyidentical to the nucleic acids of SEQ ID NOS:1-8 or nucleic acidscomprising nucleic acids substantially identical to the nucleic acids ofSEQ ID NOS:1-8, wherein substantial identity at the nucleotide leveloccurs when at least about 60% to 75% or preferably at least about 80%or most preferably at least about 90% of the nucleotides comprising thenucleic acid molecule are identical over a defined length of themolecule, while the substantially identical nucleic acids retain theability to encode for proteins having the biological function of thepolypeptides having the amino acid sequences of SEQ ID NOS:9-16.

Substantially identical nucleic acid molecules may be identified byhybridization under suitably stringent hybridization conditions.Defining appropriate hybridization conditions is within the skill of theart. See e.g. Current Protocols in Molecular Biology, Volume I. Ausubelet al., eds. John Wiley: New York N.Y., pp. 2.10.1-2.10.16, firstpublished in 1989 but with annual updating, wherein maximumhybridization specificity for DNA samples immobilized on nitrocellulosefilters may be achieved through the use of repeated washings in asolution comprising 0.1-2×SSC (15-30 mM NaCl, 1.5-3 mM sodium citrate,pH 7.0) and 0.1% SDS (sodium dodecylsulfate) at temperatures of 65-68°C. or greater. For DNA samples immobilized on nylon filters, a stringenthybridization washing solution may be comprised of 40 mM NaPO₄, pH 7.2,1-2% SDS and 1 mM EDTA. Again, a washing temperature of at least 65-68°C. is recommended, but the optimal temperature required for a trulystringent wash will depend on the length of the nucleic acid probe, itsGC content, the concentration of monovalent cations and the percentageof formamide, if any, that was contained in the hybridization solution(Current Protocols in Molecular Biology, Volume I. Ausubel et al., eds.John Wiley: New York N.Y., pp. 2.10.1-2.10.16. 1989 with annualupdating), all of which can be determined by the skilled artisan.

The present invention is further directed to purified Mint proteins orMint protein variants having enhanced abilities to modulatetranscriptional activation, and particularly the transcriptionalactivation mediated by the cytoplasmic tail of APP, relative towild-type Mint proteins. In preferred embodiments, these proteins arethose having the amino acid sequences of SEQ ID NOS:9-16. In furtherembodiments, the proteins are polypeptides encoded by the nucleic acidshaving the nucleotide sequences of SEQ ID NOS:1-8. In other embodiments,the proteins are polypeptides encoded by nucleic acids that aresubstantially identical to the nucleic acids of SEQ ID NOS:1-8, whereinsubstantial identity at the nucleotide level occurs when at least about60% to 75% or preferably at least about 80% or most preferably at leastabout 90% of the nucleotides comprising the nucleic acid molecule areidentical over a defined length of the molecule.

The present invention further provides a method of modulating thetranscriptional activation mediated by the cytoplasmic tail of APP. Inone embodiment, this method comprises introducing into a target cell anucleic acid encoding a Mint protein or a Mint protein variant havingenhanced abilities to modulate the transcriptional activation mediatedby the cytoplasmic tail of APP relative to wild-type Mint proteins.

In preferred embodiments, the nucleic acids are those having thenucleotide sequences of SEQ ID NOS:1-8. In other preferred embodiments,the nucleic acids are nucleic acids encoding the polypeptides having theamino acid sequences of SEQ ID NOS:9-16. In other embodiments, thenucleic acids are nucleic acids comprising the nucleic acids having thenucleotide sequences of SEQ ID NOS:1-8 or the nucleic acids encoding thepolypeptides having the amino acid sequences of SEQ ID NOS:9-16. Instill further embodiments, the nucleic acids are those substantiallyidentical to the nucleic acids of SEQ ID NOS:1-8 or nucleic acidscomprising nucleic acids substantially identical to the nucleic acids ofSEQ ID NOS:1-8, wherein substantial identity at the nucleotide leveloccurs when at least about 65% to 70% or preferably at least about 80%or most preferably at least about 90% of the nucleotides comprising thenucleic acid molecule are identical over a defined length of themolecule.

The nucleic acids described above may be introduced into target cellsusing a variety of vectors. These vectors include virus-based vectorsand non-virus based DNA or RNA delivery systems. Examples of potentialvirus-based gene transfer vectors include, but are not limited to, thosederived from the following nonlimiting virus types: Adenoviridae;Birnaviridae; Bunyaviridae; Caliciviridae, the Capillovirus group; theCarlavirus group; the Carmovirus group; the Caulimovirus group; theClosterovirus group; the Commelina Yellow Mottle Virus group; theComovirus virus group; Coronaviridae; the PM2 Phage group;Corcicoviridae; the Cryptic Virus group; the Cryptovirus group; theCucumovirus Virus group; the Family Φ6 Phage group; Cysioviridae; theCarnation Ringspot group; the Dianthovirus group; the Broad Bean Wiltvirus group; the Fabavirus virus group; Filoviridae; Flaviviridae; theFurovirus group; the Germinivirus group; the Giardiavirus group;Hepadnaviridae; Herpesviridae; the Hordeivirus virus group; theIllarvirus group; Inoviridae; Iridoviridae; Leviviridae;Lipothrixviridae; the Luteovirus group; the Marafivirus group; the MaizeChlorotic Dwarf Virus group; Icroviridae; Myoviridae; the Necrovirusgroup; the Nepovirus group; Nodaviridae; Orthomyxoviridae;Papovaviridae; Paramyxoviridae; the Parsnip Yellow Fleck Virus group;Partitiviridae; Parvoviridae; the Pea Enation Mosaic Virus group;Phycodnaviridae; Picomaviridae; Plasmaviridae; Prodoviridae;Polydnaviridae; the Potexvirus group; Potyvirus; Poxviridae; Reoviridae;Retroviridae; Rhabdovindae; the Rhizidiovirus group; Siphoviridae; theSobemovirus group; SSV1-Type Phages; Tectiviridae; Tenuivirus;Tetraviridae; the Tobamovirus group; the Tobravirus group; Togaviridae;the Tombusvirus group; the Torovirus group; Totiviridae; the Tymovirusgroup; and Plant virus satellites.

In preferred embodiments, the viral vectors are those derived fromretroviruses, for example Moloney murine leukemia-virus based vectorssuch as LX, LNSX, LNCX or LXSN (Miller and Rosman, Biotechniques1989;7:980-989); lentiviruses, for example human immunodeficiency virus(“HIV”), feline leukemia virus (“FIV”) or equine infectious anemia virus(“EIAV”)-based vectors (Case et al., 1999, Proc Natl Acad Sci USA96:2988-2993; Curran et al., 2000, Molecular Ther 1:31-38; Olsen, 1998,Gene Ther 5:1481-1487); adenoviruses (Zhang, 1999, Cancer Gene Ther6:113-138; Connelly, 1999, Curr Opin Mol Ther 1:565-572), for exampleAd5/CMV-based E1-deleted vectors (Li et al., 1993, Human Gene Ther4:403-409); adeno-associated viruses, for example pSub201-basedAAV2-derived vectors (Walsh et al., 1992, Proc Natl Acad Sci USA89:7257-7261); herpes simplex viruses, for example vectors based onHSV-1 (Geller and Freese, 1990, Proc Natl Acad Sci USA 87:1149-1153);baculoviruses, for example AcMNPV-based vectors (Boyce and Bucher, 1996,Proc Natl Acad Sci USA 93:2348-2352); SV40, for example SVluc (Strayerand Milano, 1996, Gene Ther 3:581-587); Epstein-Barr viruses, forexample EBV-based replicon vectors (Hambor et al., 1988, Proc Natl AcadSci USA 85:4010-4014); alphaviruses, for example Semliki Forest virus-or Sindbis virus-based vectors (Polo et al., 1999, Proc Natl Acad SciUSA 96:4598-4603); vaccinia viruses, for example modified vaccinia virus(MVA)-based vectors (Sutter and Moss, 1992, Proc Natl Acad Sci USA89:10847-10851) or any other class of viruses that can efficientlytransduce human tumor cells and that can accommodate the nucleic acidsequences required for physiologic efficacy.

Non-limiting examples of non-virus-based delivery systems that may beused according to the invention include, but are not limited to,so-called naked nucleic acids (Wolff et al., 1990, Science247:1465-1468), nucleic acids encapsulated in liposomes (Nicolau et al.,1987, Meth Enzymology 149:157-176), nucleic acid/lipid complexes(Legendre and Szoka, 1992, Pharmaceutical Res 9:1235-1242), and nucleicacid/protein complexes (Wu and Wu, 1991, Biother 3:87-95). The vectorscomprising these nucleic acids may include, but are not limited to,plasmids, cosmids, phagemids, bacmids, artificial chromosomes orreplicons.

In another embodiment, the method of modulating transcriptionalactivation comprises introducing into a target cell a Mint protein or aMint protein variant having enhanced abilities to modulatetranscriptional activation relative to wild-type Mint proteins. Inpreferred embodiments, these proteins are those having the amino acidsequences of SEQ ID NOS:9-16. In other embodiments, the proteins arepolypeptides encoded by the nucleic acids having the nucleotidesequences of SEQ ID NOS:1-8. In other embodiments, the proteins arepolypeptides encoded by nucleic acids that are substantially identicalto the nucleic acids of SEQ ID NOS:1-8, wherein substantial identity atthe nucleotide level occurs when at least about 60% to 75% or preferablyat least about 80% or most preferably at least about 90% of thenucleotides comprising the nucleic acid molecule are identical over adefined length of the molecule.

The amount of a Mint protein within a cell may be increased byintroducing the protein directly into the target cell. For example, forintroduction into a cell, the Mint protein could be incorporated into amicroparticle for uptake by pinocytosis or phagocytosis. In otherembodiments, the Mint protein could be incorporated into a liposome.Other protein-stabilizing formulations useful for the delivery ofproteins to target cells are known in the art.

The present invention also provides a method of identifying compoundsthat modulate transcriptional activation comprising contacting a cellcontaining an APP molecule modified in the C-terminal cytoplasmic tailto permit the specific transcriptional activation of a reporter gene andmeasuring the levels of reporter gene transcription in the presence andabsence of the compound, wherein increased or decreased levels ofreporter gene transcription in the presence of the compound indicatethat the compound is capable of modulating transcriptional activation.

The term APP as used herein includes naturally occurring mammalian APPand also APP that has been modified, for example in a way to facilitatemeasurement of the transcriptional activation produced by thecytoplasmic tail of APP. Naturally occurring human APP is a 695 aminoacid protein, in which the C-terminal 47 residues are designated thecytoplasmic tail. The gene encoding APP, its splice variants, andresulting nucleotide and amino acid sequences are known in the art anddisclosed for example by Kang et al., supra; Selkoe, supra; Bayer etal., 1999, supra; Haass et al., supra; and Price et al., supra, thedisclosures of which are incorporated herein by reference.

Further, APP as defined herein may include other modifications such asinsertions, deletions and substitutions provided that the functions ofability of the cytoplasmic tail or part thereof to be cleaved from theremainder of APP and translocated to the nucleus are retained.

The C-terminal cytoplasmic tail of APP may be modified to allowdetection of nuclear localization. The modification may be in any regionof the cytoplasmic tail. The modification may be at the C-terminal orN-terminal end of the tail, for example at the junction of thetransmembrane and cytoplasmic domains. In one embodiment, thecytoplasmic tail of APP is modified to include the DNA binding domainand the activation domain of the same or different heterologoustranscription factors. Heterologous as used herein means not derivedfrom a gene encoding APP. In this embodiment, nuclear localization ismeasured by determining activation of transcription of a reporter genethat is under the transcriptional control of a binding site for the DNAbinding domain. Transcription factors and their component DNA-bindingand activation domains are well known in the art.

In a preferred embodiment, the cytoplasmic tail is modified to include aheterologous DNA-binding domain such as the DNA-binding domain of theyeast transcription factor Gal4, or the bacterial LexA DNA bindingdomain. The Gal4 and LexA DNA binding domains are known in the art anddisclosed for example by Giniger et al., 1985, Cell 40:767-774 andHurstel et al., 1986, EMBO J 5:793-798. The modification may furthercontain the transcriptional activation domain of Gal4, or anotheractivator such as the viral VP16 activator, which is disclosed forexample by Stringer et al., 1990, Nature 345:783-786. In a preferredembodiment, the cytoplasmic tail of APP is modified to include Gal4 andVP16. A transcription factor module of Gal4-VP16 is described bySadowski et al., 1988, Nature 335:563-564. Accordingly, the modificationof the cytoplasmic tail may consist of a module consisting of aDNA-binding domain and a transcriptional activation domain, which may befrom the same or different sources.

The reporter gene is operably linked to a binding site for theDNA-binding protein. For example, the reporter gene may be provided inthe form of a Gal4- or LexA-dependent reporter plasmid containing areporter gene such as luciferase or chloramphenicol acetyl transferaseunder the control of a Gal4 or LexA regulatory element, respectively,such as an upstream activating sequence. Translocation of thecytoplasmic tail of APP to the nucleus results in translocation of thetranscription factor as well, resulting in activation of transcriptionof the reporter gene. Accordingly, detection of the reporter geneproduct provides an assay for nuclear localization of the cytoplasmictail of APP, and hence measures cleavage of APP. Transcriptionalactivation assays are described by Fields et al., 1989, Nature340:245-246, the disclosure of which is incorporated herein byreference. Gal4 and LexA reporter plasmids are described by Lillie etal., 1989, Nature 338:38-44 and Hollenberg et al., 1995, Mol Cell Biol15:3813-3822.

Candidate compounds that may be tested by the assays of the presentinvention include proteins, peptides, non-peptide small molecules, andany other source of therapeutic candidate compounds. The compounds maybe naturally occurring or synthetic, and may be a single substance or amixture. Screening may be performed in high throughput format usingcombinatorial libraries, expression libraries and the like. Compoundsidentified as inhibitors of the transcriptional activation mediated bythe cytoplasmic tail of APP may be subsequently tested for biologicalactivity and used as therapeutics or as models for rational drug design.

Modulation in this context is defined as little as a 5% increase ordecrease in transcriptional activation of the cytoplasmic tail of APP inthe presence of the compound relative to the level of transcriptionalactivation in the absence of the compound. In preferred embodiments, thelevel of increase or decrease greater than 10% or more preferablygreater than 20%.

Cells useful for the assays of the present invention include eukaryoticcells in which the cytoplasmic tail of APP can be translocated to thenucleus. Suitable cells include, for example, insect and mammaliancells. Preferred cells include Schneider, PC12, COS, HeLa and HEK293cells.

Cells containing APP may be cells stably or transiently transfected witha construct encoding APP as described above using methods known to thoseof ordinary skill in the art. Constructs containing chimeric genescomprising a promoter operably linked to nucleic acid encoding an APPmodified to include a module comprising the DNA-binding domains andtranscriptional activation domain of the same or different transcriptionfactor, are constructed using well-known recombinant DNA methods. Theseconstructs are co-transfected into cells with the corresponding reporterconstructs described above.

The transfected cells are contacted with the compound to be tested forits ability to modulate the transactivation mediated by the cytoplasmictail of APP. A detectable increase or decrease in transcriptionalactivation of the reporter gene is indicative of a compound thatmodulates APP-mediated transcriptional activation. In one embodiment,the cells may be contacted with the candidate compound before expressionof the modified APP is induced from an inducible promoter.

In a preferred embodiment of the present invention, human APP ismodified to include Gal4-VP16 within the cytoplasmic tail. Inparticular, Gal4-VP16 is inserted between residues 651 and 652 of APP.The modified APP is generated by means of a mammalian expression plasmidcontaining a chimeric gene encoding residues 1-651 of APP, Gal4, VP16,and residues 652-695 of APP (i.e. the cytoplasmic tail of APP) under thecontrol of a promoter (see Example #4). The plasmid may further compriseregulatory sequences, linkers, and other elements to facilitate cloning,replication, transfection and expression.

A cell comprising the modified APP is provided by transfecting a cell,preferably a mammalian cell, and most preferably a human cell, with theexpression plasmid. The cell is co-transfected with a Gal4 reporterplasmid in which luciferase mRNA is driven by multiple copies of theGal4 upstream activating sequence (UAS). When the modified APP iscleaved by γ-secretase, the cleavage product containing Gal4-VP16 entersthe nucleus and activates transcription from the Gal4 reporter plasmid.The transfected cells are contacted with a candidate compound, andluciferase expression is measured in the presence and absence of thecompound. Expression of luciferase is measured by standard assays, forexample by measuring luciferase activity using a commercially availablekit. Luciferase expression is a measure of transactivation. The compoundthat increases or decreases luciferase expression is the compound thatmodulates transcriptional activation.

In another preferred embodiment of the present invention, a cell,preferably a mammalian cell, and most preferably a human cell, isco-transfected with plasmids expressing the APP/Gal4-VP16 fusionprotein, a Gal4 reporter plasmid in which luciferase mRNA is driven bymultiple copies of the Gal4 upstream activating sequence (UAS), and aplasmid expressing one of the Mint proteins encoded by the nucleic acidsof SEQ ID NOS:1-8, the nucleic acids encoding the polypeptides havingthe amino acid sequences of SEQ ID NOS:9-16, nucleic acids comprisingthe nucleic acids having the nucleotide sequences of SEQ ID NOS:1-8 orthe nucleic acids encoding the polypeptides having the amino acidsequences of SEQ ID NOS:9-16, or nucleic acids substantially identicalto the nucleic acids of SEQ ID NOS:1-8 or nucleic acids comprisingnucleic acids substantially identical to the nucleic acids of SEQ IDNOS:1-8. The Mint protein expressed from one of these nucleic acids willinteract with the cytoplasmic tail of the APP/Gal4-VP16 fusion protein,preventing it from activating transcription of the luciferase genepresent on the reporter plasmid (see Example #7). Compounds whichpotentiate the modulatory affects of Mint on transcriptional activationmay be identified by comparing the level of expression of the luciferaseor any other suitable reporter gene from the reporter plasmid in thepresence and absence of the compound. The compound that increases ordecreases luciferase expression is the compound that potentiates themodulatory affects of Mint on transcriptional activation. Other reportergenes suitable for use in this assay are known to those of ordinaryskill in the art.

In another embodiment, the present invention provides vectors thatcontain nucleic acids encoding the Mints proteins. In preferredembodiments, the vector comprises one of the nucleic acids having thenucleotide sequences of SEQ ID NOS:1-8 operably linked to expressioncontrol sequences, e.g. a promoter. In other preferred embodiments, thevector comprises a nucleic acid encoding one of the polypeptides havingthe amino acid sequences of SEQ ID NOS:9-16 operably linked to apromoter. In other embodiments, the vector comprises nucleic acids thatare substantially identical to the nucleic acids of SEQ ID NOS:1-8operably linked to a promoter, wherein substantial identity at thenucleotide level occurs when at least about 60% to 75% or preferably atleast about 80% or most preferably at least about 90% of the nucleotidescomprising the nucleic acid molecule are identical over a defined lengthof the molecule, and wherein the nucleic acids still encode peptideshaving the biological function of the polypeptides having the amino acidsequences of SEQ ID NOS:9-16.

In another embodiment, the present invention provides vectors thatcontain nucleic acids encoding the modified APP protein. In preferredembodiments, the vector comprises a nucleic acid encoding APP operablylinked to a promoter wherein a nucleic acid module encoding aheterologous DNA binding domain of a transcription factor and atranscriptional activator of the same or a different transcriptionfactor is contained within the portion of the nucleic acid that encodesthe C-terminal cytoplasmic tail of APP. A module “within” the tailincludes embodiments in which the module is at the 5′-end or 3′-end ofthe region encoding the cytoplasmic tail. In a preferred embodiment themodule is Gal4-VP16. The vectors may further comprise regulatorysequences, linkers, and other elements to facilitate cloning,replication, transfection and expression.

The present invention further provides cells containing the foregoingvectors. The cells are eukaryotic, preferably mammalian, and mostpreferably human. Cells containing the vectors of the invention may beobtained by methods known in the art, and may be transiently or stablytransfected. The cells may also further contain a corresponding reporterplasmid as described hereinabove.

The present invention further provides kits useful for identifying acompound that modulates transcriptional activation. The kits maycomprise vectors encoding modified APP, modified Mint proteins, and thereporter gene. Alternatively, the kits may contain cells transformed byone or more of these vectors or cells suitable for transfection by thesevectors and a means for transfecting these cells. The kits may alsocomprise a means for measuring expression of the reporter gene containedin the reporter plasmid.

The present invention also provides for transgenic knockout mice forMint 1, Mint 2 and Mint 3. These animals may be useful for elucidatingthe pathophysiology of AD and for developing improved treatments forthis disease.

The following nonlimiting examples serve to further illustrate thepresent invention.

EXAMPLES

Materials and Methods

Plasmid construction. 1. Gal4-containing transactivation plasmids. Mostof the plasmids used for the transactivation experiments describedherein were reported previously (Cao and Südhof, 2001, Science293:115-120). All eukaryotic expression vectors containing Gal4 orGal4/VP16 were based on pMst (Gal4) and pMst-GV (Gal4/VP16) which arederived from the SV40 promoter-based mammalian expression vector pM(Clontech; Cao and Südhof, 2001, Science 293:115-120). In addition tothe previously described vectors, the following vectors wereconstructed: PMSt-GV-APP_(ICF) (APP_(ICF)-Gal4/VP16), generated bycloning the intracellular fragment of human APP₆₉₅ (APP_(ICF), residues652-695) into the BamHI/SalI sites of pMst-GV; pMst-GV-APP(APP-Gal4/VP16), by cloning the extracellular and TMR fragments of humanAPP₆₉₅ (APPe, residues 1-651) into the NheI site of pMst-GV-APP_(ICF);pMst-GV-APPγ (APPγ-Gal4/VP16), by cloning residues 639-651 of humanAPP₆₉₅ preceded by a methionine into the BglII/NheI sites ofPMst-GV-APP_(ICF). PMst-GV-APP_(ICF)* (APP_(ICF)*-Gal4/VP16),pMst-GV-APP* (APP*-Gal4/VP16), and pMst-GV-APPγ* (APPγ*-Gal4/VP16) weregenerated from their respective parent plasmid by site-specificmutagenesis replacing NPTY₍₆₈₄₋₆₈₇₎ with NATA₍₆₈₄₋₆₈₇₎ using theQUIKCHANGE® mutagenesis kit (Stratagene, La Jolla). pMst-GV-NRX(NRX-Gal4/VP16) was generated by cloning the intracellular fragment ofrat Neurexin 1β (NRX_(ICF), residues 414-468) into the BamHI/SalI sitesof pMst-GV, followed by cloning the extracellular and TMR fragments(NRXe, residues 1-417) into the NheI site. pMst-GV-NA(NRXe-Gal4/VP16-APP_(ICF)) was generated by cloning NRXe into the NheIsite of pMst-GV-APP_(ICF), and pMst-GV-AN (APPe-Gal4/VP16-NRX_(ICF)) bycloning the intracellular fragment of Neurexin 1β (NRX_(ICF)) into theBamHI/SalI-sites of pMst-GV, followed by cloning of APPe into the NheIsite. 2. Mint plasmids. The eukaryotic pCMV5 expression vectors forfull-length rat Mints 1, 2 and 3 have been described previously (Okamotoand Südhof, 1997, J Biol Chem 272:31459-31464; Okamoto and Südhof, 1998,Eur J Cell Biol 77:161-165). Mutations were inserted in these parentvectors by site-specific mutagenesis as described above. pCMV-Mint1-PDZ1* was constructed by mutating GV_((670,671)) to AA_((670,671)) inthe first PDZ domain's carboxylate binding loop, and pCMV-Mint 1PDZ2*was created by mutating GF_((762,763)) to AA_((762,763)) in thecarboxylate binding loop of the second PDZ domain. pCMV-Mint 1-ΔPDZ wasgenerated by introducing a stop codon after residue 659 in pCMV-Mint 1.A hydrophobic pocket of the PTB domain of Mint 1 was altered atpositions YQEF_((613,616)) to SQES_((613,616)) to generate the pCMV-Mint1-PTB* construct. pCMVmyc-Mint 1 was generated by cloning thefull-length Mint 1 coding sequence from pEGFP-Mint 1 (a gift from Dr.Anton Maximov, UT Southwestern, Dallas) into the EcoRI/KpnI sites ofpCMVmyc. To construct pCMVmyc-Mint 1-ΔNterm, the C-terminal part of Mint1 encoding residues 451-839 was cloned into the KpnI/XbaI sites ofpCMVmyc. pCMV-Mint 2-ΔPDZ was generated by introducing a stop codonafter residue 570 in pCMV-Mint 2, and pCMV-Mint 3-ΔPDZ by introducing astop codon after residue 391 in pCMV-Mint 3. 3. Other plasmids. Theeukaryotic expression vectors for CASK (Hata et al., 1996, J Neurosci16: 2488-2494) and Fe65 (Cao and Südhof, 2001, Science 293:115-120) weredescribed previously.

Antibodies. Some antibodies were described previously (Biederer andSüdhof, 2000, J Biol Chem 275:39803-39806; Biederer and Südhof, 2001, JBiol Chem 276: 47869-47876; Cao and Südhof, 2001, Science 293:115-120).The APP antibody used was a polyclonal rabbit serum (U955) raisedagainst the cytosolic, extreme C-terminal 15 residues of APP coupled tokeyhole-limpet hemocyanin. Monoclonal antibodies to Mints 1, 2 and 3,TGN 38, and EEA1 were obtained from Transduction Laboratories (LexingtonKy.); monoclonal antibodies to calnexin were obtained from Chemicon(Temecula Calif.); and monoclonal antibodies to Golgi 58K protein wereobtained from Sigma (St. Louis Mo.). Polyclonal Mint 1 antibodies weredescribed previously (P932; Okamoto and Südhof, 1997, J Biol Chem272:31459-31464), as were antibodies against Velis (T813; Butz et al.,1998, Cell 94:773-782). Polyclonal anti-myc antibodies were obtainedfrom Santa Cruz Biotechnology (Santa Cruz Calif.), and polyclonalantibodies directed against Mint 3 were obtained from AffinityBioreagents (Denver Colo.). For all Mint antibodies, specificity wasconfirmed using preparations from COS cells transfected with expressionvectors for Mints 1-3 (data not shown).

Biochemical preparations. All steps were carried out on ice or at 4° C.Rat forebrains (Pel Freez, Rogers AK) were homogenized in a pestletissue grinder using a slow speed stirrer at a tissue:buffer ratio of10% (w/v) in buffer RMP (20 mM Hepes-KOH (pH 7.4), 125 mM K-acetate, 5mM MgCl₂, 320 mM sucrose) adjusted to 1.0% TRITON® X-100 in the presenceof protease inhibitors. For preparation of membrane proteins, ratforebrains were homogenized in buffer RMP, the samples were centrifugedin an Eppendorf (Hamburg, Germany) microcentrifuge at 600 g for 10 mm toobtain the postnuclear supernatant, and membranes were pelleted in aSorvall (Kendro Laboratory Products, Newtown Conn.) S80-AT3 rotor at280,000 g for 20 mm. The membrane pellet was extracted in buffer RMPadjusted to 1.0% TRITON® X-100 using a pestle tissue grinder, andcentrifuged again at 280,000 g for 20 mm to yield the solubilizedmembrane proteins.

Peptide bead affinity chromatography. Peptides were synthesized on anABI (Applied Biosystems, Foster City Calif.) synthesizer with an addedN-terminal cysteine for coupling to SulfoLink® Beads (a 6% cross-linkedbeaded agarose matrix that has been derivatized with a 12-atom spacerarm that ends in an iodoacetyl group; Pierce, Rockford Ill.) accordingto the manufacturer's instructions at 1.0 mg peptide/ml beads. Forbinding to the APP NPTY motif, a peptide corresponding to theAPP-derived sequence CGYENPTYKFFEQMQN (human APP, residues 398-412), wasimmobilized on SulfoLink® (a 6% cross-linked beaded agarose matrix thathas been derivatized with a 12-atom spacer arm that ends in aniodoacetyl group; Pierce, Rockford Ill.) beads. For binding toPresenilin C-terminal sequences, peptides corresponding to the extremeC-termini of human Presenilin 1 (sequence CMDQLAFHQFYI; SEQ ID NO:18),human Presenilin 2 (sequence CMDTLASHQLYI; SEQ ID NO:19), or DrosophilaPresenilin (sequence CMEDLSAKQVFI; SEQ ID NO:20) were immobilized. Asnegative controls, peptides corresponding to the extreme C-terminus ofhuman HPV2 (poliovirus receptor-related protein 2; sequenceCGSLISRRAVYV; SEQ ID NO:21) and a peptide derived from the gp41glycoprotein (sequence CWFSITNWLWYI; SEQ ID NO:22) were utilized.Extracts of transfected eukaryotic cells or proteins solubilized fromrat brain were incubated with 20 μg peptide immobilized on SulfoLink®beads for 12-16 h at 4° C. under mild agitation. Binding was performedin buffer RMP adjusted to 1.0% TRITON® X-100 and 600 mM potassiumacetate. For APP competition experiments, the soluble peptidesQNGYENPTYKFFEQ (SEQ ID NO:23) or QNGYENATAKFFEQ (SEQ ID NO:24),corresponding to the native or mutated APP NPTY (SEQ ID NO:17) motif,were added during the binding incubation at the concentrations of 0.1mg/ml and 1.0 mg/ml. Bound proteins were eluted with 2% SDS.

Immunoprecipitations. HEK293 were co-transfected for APP and theindividual Mints 1, 2 or 3, respectively, and after 2 days collected inIP buffer (25 mM Hepes-KOH (pH 7.4), 125 mM K-acetate, 5 mM MgCl₂, 1.0%IGEPAL CA-630, Sigma), 10% glycerol) in the presence of proteaseinhibitors. After passing the cell suspension through a 28 gaugesyringe, the lysate was centrifuged in an Eppendorf microcentrifuge at21,000 g for 20 min and the detergent-extracted material was subjectedfor 2 h to immunoprecipitation using antibodies directed against APP(U955) or the respective pre-immune serum.

Miscellaneous biochemical procedures. SDS-PAGE and immunoblotting wasperformed as described (Laemmli, 1970, Nature 277:680-685; Towbin etal., 1979, Proc Natl Acad Sci USA 76:4350-4354). For standardimmunodetection on Western blots, enhanced chemiluminiscence (ECL;Amersham) was applied. Quantitative immunoblotting was performed usingradiolabeled ¹²⁵I secondary antibodies (Amersham Biosciences, PiscatawayN.J.), and the signals were quantitated on a PhosphorImager (MolecularDynamics, Sunnyvale Calif.) using ImageQuant software. To determinelevels of expressed Mint 1 protein, signals of cell lysates werecompared to those from known amounts of purified GST-Mint 1. Proteinconcentrations were determined using the BCA protein assay (Pierce,Rockford Ill.).

Immunocytochemistry. 1. Immunoperoxidase staining: Adult mice wereperfusion-fixed in 4% paraformaldehyde in phosphate-buffered saline(PBS) pH 7.4. 35 μm vibratome sections from brain were blocked in 10%normal goat serum containing 0.1% TRITON® X-100 (octyl phenolethoxylate) for one hour, incubated with the various primary antibodiesovernight at 4° C. and with the biotinylated secondary antibody for 1hr. Sections were processed using a VECTASTAIN® ABC Elite Kit, whichcontain avidin DH and biotinylated enzyme (Vector, Burlingame, Calif.)according to the manufacturer's instruction. The final immunosignal wasdeveloped using 3′3-diaminobenzidine tetrahydrochloride (DAB). Forimmunofluorescence labeling, primary hippocampal cells on cover slipswere fixed in situ for 10 min with absolute methanol at −20° C.,permeabilized in 0.1% saponin/PBS, and blocked in 3% milk/PBS. Theprimary incubation was carried out in blocking buffer for one hour atroom temperature. After washing with PBS, the cells were incubated withgoat anti-rabbit or goat anti-mouse secondary antibodies that werecoupled with ALEXA FLUOR® 488 and ALEXA FLUOR® 546 (fluorescent dyes;Molecular Probes, Eugene Oreg.). Labeled cells were viewed with a Leica(Leica Microsystems, Bannockburn Ill.) TCS SP2 confocal microscope or aZeiss (Carl Zeiss Optical Inc., Chester Va.) fluorescent microscope witha Hamamatsu (Bridgewater N.J.) ORCA-100 digital camera. Final imageswere processed by METAMORPH® (an integrated software and hardware systemfor the capture and analysis of microscopic or macroscopic images fromscientific grade digital and video CCD cameras; Universal Imaging,Downingtown Pa.) and ADOBE® PHOTOSHOP® imaging analysis software.

Transfections were performed at 50-80% confluency in 6-well plates usingFUGENE6™ transfection reagent (Roche, Basel, Switzerland).

Transactivation assays. PC12, COS, HeLa, and HEK293 cells wereco-transfected with three or four plasmids: a. pG5E1B-luc (HEK293 cells,HeLa cells, and COS cells=0.2-0.5 μg DNA; PC12 cells=1.0 μg); b.pCMV-LacZ (HEK293 cells, HeLa cells, and COS cells=0.05 μg DNA; PC12cells=0.5 μg DNA); c. pMst (Gal4), pMst-GV-APP (APP-GV), pMst-GV (GV),pMst-GV-APPct (APPct-GV), pMst-APPct (APPct-Gal4), pMst-GV-APP*(APP*-GV), pMst-GV-APPct* (APPct*-GV), pMst-APPct* (APPct* -Gal4),pMst-GV-APP (APP-GV), pMst-GV-NRX (NRX-GV), pMst-G V-NA (NRXe-GV-APPc),pMst-GV-AN (APPe-GV-NRXc)(HEK293, HeLa, and COS cells=0.1-0.3 μg DNA;PC12 cells 1.0 μg DNA). Where indicated, a fourth plasmid wasco-transfected: pcDNA3.1-PS2D366A (kind gift of Dr. C. Haass, Munich);pCMV-Mint1; pCMV-Mint 1-PDZ1*; pCMV-Mint 1-PDZ2*; pCMV-Mint 1-PTB*;pCMVmyc-Mint 1; pCMVmyc-Mint 1-ΔNterm; pCMV-Mint 1-ΔPDZ; pCMV-Mint 2;pCMV-Mint 2-ΔPDZ; pCMV-Mint 3; pCMV-Mint 3-ΔPDZ; pCMV5-Fe65; pCMV-CASK(HEK293, HeLa, and COS cells=0.1-0.3 g DNA or as specified in thediscussion of each individual experiment; PC12 cells=0.5 g DNA). Fornegative controls, the expression vector pCMV5 was used without insert.Cells were harvested 48 hr post-transfection in 0.2 ml/well reporterlysis buffer (Promega, Madison Wis.), and their luciferase andβ-galactosidase activities were determined with the Promega luciferaseassay kit using a chemiluminescence reader (LUCY2, Anthos Labtec,Austria) and the standard O-nitrophenyl-D-galacto-pyranoside (Sigma, St.Louis Mo.) method, respectively. The luciferase activity wasstandardized by the β-galactosidase activity to control for transfectionefficiency and general effects on transcription, and further normalizedfor the transactivation observed in cells expressing Gal4 alone whereindicated. Values shown are averages of transactivation assays carriedout in duplicate, and repeated at least three times for each cell typeand constructs. All constructs were assayed in three or four cell lines,but usually only representative results for one cell line are shown.

Example 1

Comparison of the binding of Mints/X11 1, 2, and 3 to APP. Previousresults have separately examined the ability of Mints to bind to APP orto presenilins, and to stabilize APP in co-transfected cultured cells(Borg et al., 1996, Mol Cell Biol 16:6229-6241; McLoughlin and Miller,1996, FEBS Lett 397:197-200; Zhang et al., 1997, EMBO J. 16: 6141-6150;Borg et al., 1998a, J Biol Chem 273:14761-14766; Sastre et al., 1998, JBiol Chem 273:22351-22357; Lau et al., 2000, Mol Cell Neurosci16:557-565; Okamoto et al., 2001, Eur J Neurosci 12:3067-3072). Theseexperiments established a potentially important connection betweenMints, presenilins, and APP, but the relative activities of the threeMints and the generality of these putative targets were not analyzed.

As an initial step towards understanding the common versus uniqueproperties of Mint isoforms, a comparison of the binding of differentMints to APP and presenilin, and their effect on APP in transfectedcells, was performed. Since these and subsequent experiments criticallydepended on the specificity of the Mint antibodies used, a studyvalidating the specificity of these antibodies was first performed usingtransfected COS cells that express individual Mints. As shown in FIG. 1,antibodies used for the respective Mint isoforms bound specifically toprotein extracts prepared from COS cells transfected with a controlvector (lane 1) or Mint 1-3 expression vectors (lanes 2-4).Immunoblotting was performed using monoclonal antibodies against Mints 1and 2, and polyclonal antibodies against Mint 3. Fractions were alsoanalyzed by inmmunoblotting for the negative control proteins RabGDP-dissociation inhibitor protein (GDI), a soluble protein, and forsynaptophysin 1 (Syp), a membrane protein of synaptic vesicles. Thisstudy confirmed the mono-specificity of the antibodies, which could thusbe applied to detect each Mint isoform separately in complex solutionscontaining multiple Mints, such as brain homogenates.

Affinity chromatography of rat brain proteins on the immobilizedcytoplasmic tail of APP was then used to examine the binding ofendogenous Mints to APP. Proteins from rat forebrain homogenates (lane5) were bound to an immobilized cytoplasmic peptide derived from APP inthe absence of added soluble peptide (lane 6), or in the presence of 0.1or 1.0 mg/ml of a soluble peptide containing the wild-type sequence ofthe APP cytoplasmic tail (lanes 7 and 8), or of a APP tail peptidepoint-mutated in the NPTY binding sequence for Mints (lane 9). As afurther control, binding of Mints to a control column with animmobilized peptide derived from gp41 was examined (lane 10). Bindingwas performed at 600 mM salt. The samples tested in lanes 6-10 areproteins bound to beads after the respective binding and peptidecompetition experiments. Recovery of the three Mints from the brainhomogenates was quantitated with ¹²⁵I-labeled secondary antibodies, asshown in FIG. 1A, lanes 5-10. All three Mints tightly bound to APP (FIG.1A, lane 6), and binding was inhibited by high concentrations of thecorresponding wild-type but not mutant APP tail peptide (FIG. 1A, lanes7-9). As shown in FIG. 1B, quantitation revealed that Mints from thebrain homogenate were recovered efficiently on the affinity column withyields of 25% to 90%; for example, the immobilized APP tail extractedalmost all of the brain Mint 3, and even 1 mg/ml of competing peptidewas unable to completely block it from binding to the column. Incontrast, only 25% of brain Mint 1 were bound. No Mint binding to acontrol peptide was observed (FIG. 1A, lane 10).

To test if Mints interact with APP in vivo, the levels of thetransfected APP were measured by quantitative immunoblotting using¹²⁵-labeled secondary antibodies (FIGS. 2A and 2B). As a control, thegene for Fe65, a protein that also binds to the cytoplasmic tail of APP(Fiore et al., 1995, J Biol Chem 270:30853-30856), was transfected.Under control conditions (lane 1), little APP₆₉₅ is expressed in thecells because the predominant splice variants in peripheral tissuesinclude the Kunitz domain (Kitaguchi et al., 1988, Nature 331:530-532;Tanzi et al., 1988, Nature 331:528-30). The migration position of theendogenous APP containing the Kunitz domain and the transfected APP₆₉₅lacking this domain are indicated on the right of the panel. Thequantities of loaded lysates were normalized to equal amounts oftransfected cells based on the activity of co-transfectedβ-galactosidase.

All three Mints dramatically increased APP levels (up to three-fold;FIG. 2, lanes 3-5), extending previous observations that Mint 1stabilizes transfected APP and increases its steady-state levels (Borget al., 1998a, J Biol Chem 273:14761-14766; Sastre et al., 1998, J BiolChem 273:22351-22357). As a control, Fe65 (which also binds to APP)slightly decreased the steady-state levels of transfected APP, althoughthis was not necessarily a specific effect since co-transfection of anyprotein usually decreases expression because it dilutes thetranscription/translation machinery. The stabilization of APP in thetransfected cells could be due to a direct or indirect interaction ofMints with APP. The levels of endogenous APP do not change significantlybecause most of the cells are not transfected, and thus are not exposedto the transfected Mints.

Although the affinity chromatography experiments already suggested adirect interaction (FIG. 1), this question was further examined by theco-immunoprecipitation of Mints with APP from the transfected cells(FIG. 2C). In these studies, APP₆₉₅ and Mints 1, 2, or 3 wereco-expressed in transfected HEK293 cells (“Start”; lane 1). Cellularproteins were then immunoprecipitated with an antibody to the C-terminusof APP (“IP APP”; lane 2), or a control antibody (“IP Control”; lane 3).Samples were analyzed by immunoblotting with monoclonal antibodies tothe indicated proteins. The double band for Mint 2 is probably due to ahypersensitive proteolytic site in the N-terminus of Mint 2. Fordetection of APP, the quantity loaded in lanes 2 and 3 was 5-fold lessthan for detection of Mints. Indeed, Mints 1, 2 and 3 wereco-immunoprecipitated with antibodies to APP, but not with controlantibodies (FIG. 2C), supporting the notion that Mints directly bind toAPP.

Example 2

Binding of Mints to presenilins. The potential interaction of Mints withpresenilins was examined using the same affinity chromatography approachemployed for APP binding. These studies showed that all three Mintsbound to the cytoplasmic C-terminal sequence of presenilin 1 (FIG. 3Aand data not shown), in agreement with previous observations (Lau etal., 2000, Mol Cell Neurosci 16:557-565). Binding was specific becauseMints were not bound to control beads, and control proteins such as GDIand synaptophysin were not retained on the presenilin 1 column (data notshown). However, quantitations revealed that presenilin binding differedamong Mints in (FIG. 3B, and data not shown). 14-16% of endogenous Mints1 and 3 from brain were recovered on the presenilin column, but only 3%of Mint 2 was bound.

Mints bind to the cytoplasmic tail of APP via an interaction of the MintPTB domain with the NPTY sequence in APP (Borg et al., 1996, Mol CellBiol 16:6229-6241; McLoughlin and Miller, 1996, FEBS Lett 397:197-200;Zhang et al., 1997, EMBO J. 16: 6141-6150). Presenilin 1 does notinclude an NPxY sequence but feature a C-terminal sequence thatresembles that of neurexins which bind to the PDZ domains of Mints(Biederer and Südhof, 2000, J Biol Chem. 275:39803-39806), suggestingthat the binding of Mints to presenilin 1 may be mediated by one or bothMint PDZ domains.

To test if other PDZ domain proteins also bind to the C-terminalsequence of presenilin 1, the presenilin binding of PSD95, an abundantcomponent of the postsynaptic density that contains three PDZ domains(Cho et al., 1992, Neuron 9: 929-942; Kistner et al., 1993, J Biol Chem268: 4580-4583), and of Velis, a family of three proteins that contain asingle PDZ domain (Butz et al., 1998, Cell 94:773-782), was examined.Both PDZ domain proteins were bound; whereas binding of PSD95 was weak,Velis were captured to the same extent as Mints 1 and 3 (data notshown).

The fact that multiple unrelated PDZ domain proteins bind to theC-terminal sequence of presenilin 1, but various Mints exhibit largedifferences in binding, raised concerns about the specificity of theinteraction of Mints with presenilins, prompting a further examinationof Mints binding specificity to presenilin.

The C-terminal presenilin 1 sequence is conserved in vertebratepresenilin 2 and in Drosophila presenilin. C-terminal peptides from allof these presenilins captured Mint 1, whereas control peptides did not,suggesting that all of these presenilins potentially interact withPDZ-domain proteins (FIG. 3B).

To identify which of the two PDZ domains in Mints binds to presenilins,we mutated the PDZ1 and PDZ2 domains of Mint 1 separately or together,or deleted them both. Presenilin binding assays with wild-type andmutant Mint proteins produced in transfected COS cells revealed thatmutations in each of the two Mint 1 PDZ domains resulted in anapproximately two-fold decrease in binding (FIG. 3C). Mutations in bothPDZ domains or deletion of both PDZ domains almost completely abolishedbinding. Together these data suggest that each of the two Mint PDZdomains individually binds to presenilins in vitro.

Example 3

Localization of endogenous Mints in neurons. In vertebrates, Mints 1 and2 are only detectable in brain, whereas Mint 3 is ubiquitously expressed(Okamoto and Südhof, 1997, J Biol Chem 272:31459-31464; Okamoto andSüdhof, 1998, Eur J Cell Biol 77:161-165). To study the localization ofMints, rat brain sections were stained with antibodies to Mints 1 and 2.Mint 3 could not be investigated because the available antibodies werenot suitable for immunocytochemistry.

Abundant labeling of the neuronal cell bodies, with less staining of thedendrites, was observed (FIG. 4, A, B, D, and E; see also McLoughlin etal., 1999, Neurosci 11:1988-1994). APP exhibited a very similardistribution (FIG. 4, C and F). Notably, nuclei were not labeled.Staining throughout the neuropil was detected that was weaker than thecell body staining, indicating that low levels of Mints may be presentat synapses as suggested by Okamoto and colleagues. See Okamoto et al.,2000, Eur J Neurosci 12:3067-3072. FIG. 4, D, E and F are enlargedimages of CA3 regions from FIG. 4, A, B and C. Antibodies against Mint 1were polyclonal, and monoclonal against Mint 2. The polyclonal APPantibody used is directed against a C-terminal epitope in thecytoplasmic APP tail which is liberated upon γ-cleavage. Scale bars inFIG. 4C (for FIG. 4, A-C)=0.5 mm; in FIG. 4F (for FIG. 4, D-F)=0.1 mm.

To examine the subcellular distribution of Mints, immunofluorescencestaining of cultured hippocampal neurons was performed (FIGS. 5 and 6).Mints 1 and 2 were predominantly localized in a perinuclear compartmentwhere they almost completely overlapped (FIG. 5, A-C). Double labelingwith antibodies to synapsins as a presynaptic marker failed to detecthigh levels of Mints in synapses (FIG. 5, D-F, and data not shown). APPwas largely co-localized with Mints (FIG. 5, G-L). Compared to Mints,more APP appeared to be present in neurites, suggesting that at steadystate, Mints are more concentrated in the perinuclear compartments thanAPP.

In the next set of experiments, cultured hippocampal neurons weredouble-labeled with antibodies to marker proteins to identify theperinuclear compartment containing Mints (FIG. 6). These studiesdemonstrated that Mints co-localize best with TGN38 (FIG. 6, A-C, anddata not shown), a marker of the trans-Golgi complex (Luzio et al.,1990, Biochem J 270:97-102). A similar localization, but not quite asprecise, was observed with the 58K Golgi protein (FIG. 6, D-F, and datanot shown), a peripheral membrane protein that is enriched in, but alsofound outside of, the trans-Golgi complex (Bloom and Brashear, 1989, JBiol Chem 264:16083-16092).

By contrast, double labeling of neurons with antibodies to Mint 1 or 2and the endoplasmic reticulum protein calnexin (Wada et al., 1991, JBiol Chem 266:19599-19610) failed to detect an overlap in localization(panels G-I). Similarly, the early endosomal protein EEA1 (Mu et al.,1995, J Biol Chem 270:13503-13511) exhibited a different stainingpattern (panels J-L). Together these data support the conclusion that inmature neurons in situ (FIG. 4) and in culture (FIGS. 5 and 6), Mints 1and 2 are co-localized in the trans-Golgi complex, a localizationconsistent with a role in APP trafficking (Borg et al., 1998a, J BiolChem 273:14761-14766; Sastre et al., 1998, J Biol Chem 273:22351-22357)and in directing proteins out of the trans-Golgi apparatus towardsdefined plasma membrane domains (Rongo et al., 1998, Cell 94:751-759;Whitfield et al., 1999, Mol Biol Cell 10:2087-2100). Thus at steadystate, Mints exhibit a localization that is similar to that of APP butnotably distinct from that of either CASK or Munc18-1.

Example 4

A transactivation assay of APP cleavage. A function for the cytoplasmictail of APP in activating transcription has recently been described,wherein this protein forms a protein complex with Fe65 (Cao and Südhof,2001, Science 293:115-120). The transcriptional activation observed inthese assays could potentially be used to measure APP cleavage, butdepends on Fe65 which binds to the same NPTY sequence of APP as Mints(which, however, do not activate transcription; Cao and Südhof, 2001,Science 293:115-120).

In order to test the potential function of Mints in APP cleavage andsignaling, a variant of the assay was employed that allows monitoringAPP cleavage independent of Fe65 binding. For this purpose, both Gal4and VP16 were introduced into the cytoplasmic tail of APP₆₉₅ at thecytoplasmic boundary of the TMR. This assay differs from the originalassay (Cao and Südhof, 2001, Science 293:115-120) in that the powerfulviral transcriptional activator VP16 (Sadowski et al., 1988, Nature335:563-564) is introduced together with the yeast DNA binding proteinGal4 into APP. Thus transactivation by APP-Gal4/VP16 is independent ofthe binding of cellular transcriptional activators, but can only occurafter APP is cleaved by γ-secretase and the released cytoplasmic tailfragment moves into the nucleus.

DNA encoding the APP-Gal4/VP16 fusion protein was transfected into PC12,HEK293, COS, or HeLa cells, and transactivation of transcription from aco-transfected Gal4-dependent reporter plasmid encoding luciferase wasmeasured. As a negative control, Gal4 was used alone without VP16 or APP(see Cao and Südhof, 2001, Science 293:115-120), and as a positivecontrol, Gal4/VP16 without APP was used. In all experiments, cells wereco-transfected with a constitutive β-galactosidase expression vector inorder to control for transfection efficiency and to rule out directeffects of transfected proteins on transcription. Furthermore, in allcases expression of transfected proteins was verified by immunoblotting.

The bar diagram in FIG. 7A shows results from Gal4-transactivationassays in PC12 cells that were co-transfected with a Gal4-dependentluciferase reporter plasmid (to measure transactivation using luciferaseexpression), a β-galactosidase control plasmid (to normalize fortransfection efficiency), and the test plasmids identified by numbersbelow the bars. The domain structures of the proteins encoded by thetest plasmids are shown schematically in FIG. 7B (APP_(e)=extracellularsequences of APP; APP_(ICF)=cytoplasmic sequences of APP). Constructsmarked with an asterisk (APP*-GV, APP_(ICF)*-GV, and APP_(ICF)*-Gal4)contain a point mutation in which the NPTY sequence in the cytoplasmictail of APP is replaced by NATA. Transfected cells were harvested twodays after transfection, luciferase and β-galactosidase activities weredetermined, and the luciferase activity was normalized for theβ-galactosidase activity to control for transfection efficiency asdescribed in the Materials and Methods. The β-galactosidase-normalizedluciferase activity is expressed in relation to the activity of cellsco-transfected with Gal4 alone. Data shown are from a representativeexperiment repeated multiple times in PC12 cells and in COS, HEK293, andHeLa cells with similar results. Abbreviations: GV, Gal4/VP16 module;APP_(ICF), intracellular fragment of APP; APP_(γ), γ-secretase cleavageproduct of APP.

In all cell types tested, full-length APP-Gal4//VP16 (APP-GV)transactivated Gal4-dependent transcription almost as strongly asGal4/VP16 alone (˜500-2,000 fold activation over Gal4 alone, dependingon cell type), suggesting that cleavage of APP to release theintracellular fragment was not the major rate-limiting step (FIG. 7A,Constructs 1-3). A chimeric protein in which Gal4/VP16 was fused to theisolated cytoplasmic tail of APP (APP_(ICF)-GV) was an even more potenttransactivator than Gal4/VP16 alone or full-length APP-Gal4/VP16 (˜4,000vs.˜500-2,000 fold activation; FIG. 7A, Construct 5). Addition of the 12hydrophobic residues from the TMR that are present in the initialγ-cleavage product had no inhibitory effect on transactivation, butinduced a moderate stimulation (FIG. 7A, Construct 6). In contrast, thecytoplasmic APP tail containing only Gal4 without VP16 (APP_(ICF)-Gal4)was inactive when Fe65 was not co-transfected (<5 fold activation; FIG.7A Construct 7; Cao and Südhof, 2001, Science 293:115-120). Cleavage ofAPP does not appear to require binding of endogenous cellular proteinsto the NPTY tail sequence (residues 684-687 of APP₆₉₅) since mutation ofNPTY to NATA had no effect on transactivation. Specifically, no effectof this mutation was observed with either full-length APP or thecytoplasmic APP tail fused to Gal4/VP16 (FIG. 7A, Constructs 4 and 7).

Example 5

APP sequences required for cleavage. The assay described above utilizestransfected cells that overexpress the respective test proteins, raisingthe concern that transactivation by APP-Gal4/VP16 may be caused bynon-specific proteolysis of the APP-Gal4/VP16 fusion protein instead ofspecific α-/β- and γ-cleavage. To address this concern, assays wereperformed to examine determine if specific sequences of APP wererequired for transactivation by the embedded Gal4/VP16 module (FIG. 8).

The bar diagram in FIG. 8A shows results of Gal4/VP16-transactivationassays obtained with the constructs that are schematically displayed andidentified by numbers below the diagrams. APP-Gal4/VP16 proteins thatcontain all APP sequences (construct 3) or lack the intracellular(construct 4) or the extracellular and transmembrane sequences(construct 5) were analyzed as described in the previous study above.Gal4 (construct 1) and Gal4/VP16 (construct 2) were used asstandardization controls to establish the background and maximalresponse, respectively. Transactivation was measured by constructs inwhich Gal4/VP16 is placed in all possible combinations into the contextof the extra- and intracellular sequences of APP (APP_(e) andAPP_(ICF)=extracellular and cytoplasmic sequences of APP, respectively)or neurexin 1β (NRX_(e) and NRX_(ICF)=extracellular and cytoplasmicsequences of neurexin, respectively). Gal4/VP16 constructs weretransfected without (−) and with (+) the dominant negative presenilin 2expression vector. All bar diagrams exhibit representative experimentsin the cell types identified in A. Experiments were carried out withtest plasmids co-transfected with a Gal4 luciferase reporter plasmid anda β-galactosidase control plasmid as described in the previousexperiment. In A, transactivation as measured byβ-galactosidase-normalized luciferase activity is expressed relative tothe activity of Gal4 alone, whereas in B, transactivation as measured byluciferase activity is shown in arbitrary units only normalized toβ-galactosidase activities.

In these studies, the cytoplasmic tail of APP was first removed togenerate a “tailless” APP-Gal4/VP16 fusion protein in which theextracellular sequences and the TMR of APP were linked to intracellularGal4/VP16 followed by a stop codon (APPe-GV). The tailless APP-Gal4/VP16protein was almost completely inactive in transactivation assayscompared to either Gal4/VP16 alone or to APP-Gal4/VP16 or to APPγ-GVwhich represents the initial γ-secretase cleavage product (FIG. 8A,Constructs 2-5). This suggests that Gal4/VP16 is not released fromAPP-Gal4/VP16 by non-specific degradation, and that the tail of APP isrequired either for recognition by the APP cleavage enzymes, or fortrafficking of APP to the cleavage compartments.

To differentiate between these two possibilities, we inserted Gal4/VP16into the cytoplasmic tail of neurexin 1β (NRX-GV). Neurexin 1β isexpressed on the neuronal cell-surface similar to APP, but is not knownto be processed by proteolytic cleavage (Ushkaryov et al., 1992, Science257:50-56). Neurexin 1β-Gal4/VP16 was nearly inactive in transactivationassays in contrast to APP-Gal4/VP16 (FIG. 8B, Construct 3 vs. Construct6), suggesting that Gal4/VP16-mediated transactivation requires specificsequences in the APP molecule. To identify these sequences, chimericGal4/VP16-fusion proteins containing either extracellular APP- andintracellular neurexin 1β-sequences, or vice versa, were constructed. Afusion protein composed of the extracellular sequences and TMR of APPcoupled to intracellular Gal4/VP16 and the cytoplasmic tail of neurexin1 (APP_(e)-GV-NRX_(ICF)) strongly transactivated transcription (FIG. 8B,Construct 8). In contrast, the reverse fusion protein of theextracellular neurexin sequences and the neurexin TMR with thecytoplasmic APP sequences (NRX_(e)-GV-APP_(ICF)) was inactive (FIG. 8B,Construct 7). These experiments demonstrate that the extracellularsequences of APP are essential for proper cleavage, in agreement withresults of Struhl and Adachi, 2000, Mol Cell 6:625-36.

Although the intracellular sequences of APP are also essential for itsprocessing (see “tailless” mutant, FIG. 8A), they can be functionallyreplaced by the intracellular fragment of a plasma membrane protein likeneurexin that exhibits no sequence similarity with APP, and inparticular does not contain an NPxY sequence (Ushkaryov et al., 1992,Science 257:50-56). This may indicate a role of the APP tail intrafficking to a cleavage compartment at the plasma membrane, or derivedfrom the plasma membrane.

Finally, to test if presenilins are involved in transactivation byAPP-Gal4/VP16, a plasmid encoding a dominant negative mutant ofpresenilin 2 (Steiner et al., 1999, J Biol Chem 274:28669-28673) wasco-transfected along with a plasmid encoding APP-Gal4/VP16 (FIG. 8B).Transactivation of Gal4-dependent transcription by full-lengthAPP-Gal4/VP16 was inhibited by the presenilin 2 mutant, whereas thesmall amount of residual transactivation observed with neurexin1β-Gal4/VP16 was insensitive to presenilin 2 (FIG. 8B, Construct 3 vs.Construct 6). As expected, mutant presenilin 2 also potently inhibitedtransactivation by the fusion protein of the extracellular domain of APPwith intracellular neurexin sequences (FIG. 8B, Construct 8), but had noeffect on the residual transactivation observed with the reverse fusionprotein containing the extracellular domain of neurexin 1β coupled tothe cytoplasmic tail of APP (FIG. 8B, Construct 7).

Example 6

Mint 1 inhibits transactivation mediated by APP-Gal4/VP16. Thetransactivation assay was next used to study the effects of Mints onAPP. A constant amount of wild-type or mutant APP-Gal4/VP16 plasmid (100ng DNA/well) was co-transfected with the indicated amounts of Mint 1expression vector into HEK293 cells. Transactivation and Mints levels inthe cells were quantified in the same samples as described in theMaterials and Methods. Co-transfection of Mint 1 strongly inhibitedtransactivation mediated by APP-Gal4/VP16 (FIG. 9A). This inhibition wasabolished by mutation of the NPTY sequence in the cytoplasmic tail ofAPP-Gal4/VP16, consistent with the notion that direct binding of Mint 1to APP is required.

Comparison of the activity of the three Mint isoforms in thetransactivation assay demonstrated that Mints 1 and 2 potently inhibitedtransactivation by APP-Gal4/VP16, whereas Mint 3 had no significanteffect (FIG. 9B). In these studies, Mint amounts in transactivationassay samples were quantified on immunoblots and are expressed as afraction of the amount observed with the maximal amount of DNAtransfected to control for the non-linearity of the relation betweentransfected DNA and expressed protein. Immunoblotting confirmed that allthree Mints were expressed at high levels in the co-transfected cells(FIG. 9C).

Since all three Mints bind to APP in vitro and in transfected cells(FIGS. 1 and 2), the selective inability of Mint 3 to inhibittransactivation was surprising. To gain insight into the mechanism bywhich Mint 1 inhibits APP-Gal4/VP16 mediated transactivation, and tounderstand why Mint 3 has no effect, a series of Mint mutants wereexamined. In these studies, HEK293 cells were co-transfected with aconstant amount of APP-Gal4/VP16 expression vector and reporterplasmids, and increasing amounts of expression vectors expressingwild-type Mint 1 or mutants of Mint 1 carrying point mutations in thefirst or second PDZ domains (Mint 1 PDZ1* or PDZ2*, respectively) orlacking both C-terminal PDZ domains (Mint 1 ΔPDZ). Transactivation andMint 1 amounts in the cells were then quantified in the same samples asdescribed in the Materials and Methods. All transactivation levels inthe experiments reported in FIGS. 10A and 10B are normalized for theamount of transactivation observed under control conditions.

These studies showed that an inactivating point mutation in the firstPDZ domain of Mint 1 or deletion of both PDZ domains that were studiedabove in the presenilin-binding experiments (FIG. 3) dramaticallyincreased the inhibition of transactivation by Mint 1 (FIG. 10A). Incontrast, the second PDZ domain point mutation did not alter theinhibitory effect of Mint 1.

Since the various Mint 1 mutants exhibited different expression levels,the relative amounts of expressed protein were quantified using¹²⁵I-labeled secondary antibodies. These quantitations showed that thePDZ-domain deletion mutant was approximately 10 times more potent thanwild-type Mint 1 (FIG. 10A). A Mint 3 mutant with a PDZ-domain deletionwas also tested; this mutant was completely inactive in the assaysimilar to wild-type Mint 3 (FIGS. 10B, C). Control transfections showedthat the various Mint 1 proteins did not inhibit general transcription,but specifically impaired transactivation by APP-Gal4/VP16 (data notshown).

The results of FIG. 10 suggest that Mint 1 binding to APP may couple itto another protein which binds to the first PDZ domain, implying amongothers that the two PDZ domains of Mint 1 are not equivalent. To examinethis further, the effect of mutating the PTB domain of Mint 1 on itsinhibitory activity was examined. Based on the available structuralinformation (Zhang et al., 1997, EMBO J. 16:6141-6150), a mutant Mint 1PTB* was designed in which critical residues involved in binding to theNPTY sequence of APP were altered. Specifically, YQEF₍₆₁₃₋₆₁₆₎ wasmutagenized to SQES₍₆₁₃₋₆₁₆₎. In this study, the same DNA amount ofcontrol vector or Mint 1 vector or the vectors encoding the indicatedMint 1 mutants was co-transfected into HEK293 cells with theAPP-Gal4/VP16 and reporter plasmids, and transactivation was determinedas described in the Materials and Methods. As shown in FIG. 11A, the PTBdomain mutation decreased inhibition of transactivation, but did notabolish it, possibly because the mutant PTB domain retained residualbinding activity for APP. Only the combination of the PTB domainmutation with the point mutation in the first PDZ domain of Mint1 orwith the deletion of both PDZ domains abolished its inhibitory effect ontransactivation (FIG. 11A).

Conversely, after deletion of the isoform-specific N-terminal residuesof Mint 1 (the sequences that are N-terminal to the PTB and PDZ domainsand account for 451 of the 839 residues of Mint 1; Okamoto and Südhof,1997, J Biol Chem 272:31459-31464), the specific inhibitory activity ofMint 1 was also increased significantly (FIG. 11B). In these studies,the same amounts of myc-tagged full-length Mint 1 or an N-terminallytruncated Mint 1 mutant containing only the PTB- and PDZ-domains wereco-transfected with APP-Gal4/VP16 and reporter constructs into HEK293cells, and their specific inhibitory activity on transactivation wasdetermined. To exclude an effect of the myc-epitope, the specificactivity of wild-type and myc-tagged Mint1 was compared.

Expressed proteins were detected using antibodies against the mycepitope. The expression level of the N-terminal Mint 1 deletion mutantwas low, suggesting that it may be partially cytotoxic (FIG. 11C).

Example 7

Effect of Mints on transactivation by the intracellular fragment of APP.A possible explanation for the effects of Mints on transactivation byAPP-Gal4/VP16, based on the binding of Mints to APP via their PTB domainand to presenilins via their PDZ domains (FIGS. 1-3) would be that theMints 1 and 2 interfere with γ-cleavage of APP. However, the facts thatMint 3 also binds to APP and presenilins better than Mint 2 but does notinhibit transactivation, and that both Mint 1 PDZ domains bind topresenilin 1 in vitro but only the first PDZ-domain is involved in theinhibition of transactivation argue against this hypothesis. Analternative explanation for the transactivational inhibition is thatMints 1 and 2 act on the APP cytoplasmic tail after it has been releasedby γ-cleavage.

To differentiate between these explanations, we measured the effect ofMints on transactivation by the “precleaved” cytoplasmic tail of APPfused to Gal4/VP16 (APP_(ICF)-GV). To distinguish specific, i.e.binding-dependent, effects from non-specific effects, a mutantcytoplasmic tail of APP that was unable to bind to Mints (APP_(ICF)*-GV)was also examined in the same experiments. In addition, Mints werecompared to Fe65 as another APP-binding protein, and to CASK as anunrelated control (FIG. 12). In these studies, constant amounts ofplasmids encoding APP_(ICF)-GV or the NPTY₍₆₈₄₋₆₈₇₎ to NATA₍₆₈₄₋₆₈₇₎mutant APP_(ICF)*-GV were co-transfected into HEK293 cells together withreporter plasmids and expression vectors for the indicated proteins.

Mint 1 expression significantly inhibited transactivation mediated bythe cytoplasmic tail of APP fused to Gal4/VP16; this inhibition was onlyobserved when the APP tail contained a normal Mint binding sequence(FIG. 12). A similar inhibition was detected with Mint 2 (data notshown). Mint 3, by contrast, had no significant effect, in agreementwith the results obtained with full-length APP-Gal4/VP16 (FIGS. 9B and10B). Fe65 enhanced transactivation, again only when the NPTY sequencein the cytoplasmic tail was intact (FIG. 12), consistent with theoverall function of Fe65 in stimulating transcription (Cao and Südhof,2001, Science 293:115-120). CASK used as a negative control had noeffect on transactivation. Identical effects of both Mints 1 and 3,their PDZ truncation mutants, and Fe65 were observed whentransactivation was assayed by a construct that mimicked the initialγ-cleavage product of APP, i.e. that contained 12 hydrophobic residuesfrom the TMR preceding the cytoplasmic tail of APP (data not shown).These results demonstrate that Mint 1 inhibits transactivationdownstream of APP cleavage, and that this effect is highly specific forneuronal vs. ubiquitous Mints.

Example 8

Generation of Mint 1, Mint 2 and Mint 3 KO mice. The strategies used forthe creation of the Mint 1, Mint 2 and Mint 3 KO mice are shown in FIG.14, panels A-C, respectively. Briefly, genomic clones encoding exons ofthe Mint 1, 2 and 3 genes were mapped and inducible targeting vectorswere constructed. In these inducible targeting vectors, an exon of theMint gene was flanked by loxP sites, and a neomycin gene cassette wasintroduced which was flanked by flp sites for positive selection. Anegative selection marker diphtheria toxin (DT) was placed at the end ofthe genomic sequences for negative selection. Embryonic stem (ES) cellswere electroporated with Mint 1, 2 and 3 inducible targeting vectors andsubjected to positive and negative selection. Double-resistant cloneswere screened by Southern analysis to verify proper targeting.

All three inducible targeting vectors could be successfully employed togenerate recombinant ES cells. Injection of these cells intopseudopregnant female hosts led to the production of chimeric offspringcarrying the mutant allele, which was subsequently shown to betransmitted through the germline. Crossing of the heterozygous animalsled to the production of null mutant knockout mice. As shown for Mint 1KO mice in FIG. 15, Southern blot analysis was employed to confirm thepresence of the wild-type or mutant allele.

Mint 1 null mutant knockout mice are viable and fertile. Althoughincompletely characterized at present, they show no obviousabnormalities other than that they weight comparatively less than theirage-matched, wild-type littermates. No changes in protein expression inMint 1-associated proteins such as munc18, CASK, velis, neurexins andAPP were observed. There were no abnormalities in excitatory synaptictransmission or plasticity, but an increase in the magnitude ofpaired-pulse depression was noted at inhibitory synapses in the Mint 1null mutant knockout mice, as compared to wild-type littermates, suggestthat these animals may exhibit some change in GABA release frominhibitory terminals. A second group has also recently reported thecreation and characterization of a transgenic Mint-1 knockout mouse. SeeMori et al., Neurosci Res. 2002; 43:251-257.

Null mutant knockout mice for Mint 2 and Mint 3 are also viable andfertile. Phenotypic characterization of these animals is in progress.

The foregoing data provide a model for the interaction between proteinsof the Mint family and the cytoplasmic tail of the APP wherein bindingof Mint 1 or 2 to the cytoplasmic tail of APP inhibits thetranscriptional activation otherwise mediated by this protein.

All references cited herein are incorporated herein in their entirety.

1. A method of identifying compounds that modulate transcriptionalactivation in a target cell comprising: a contacting a target cell witha test compound, wherein the target cell comprises (i) a first nucleicacid encoding an amyloid-β precursor protein (APP) fusion proteincomprising a modified cytoplasmic tail of the AAP, protein wherein thecytoplasmic tail is modified to include a heterologous DNA-bindingdomain of a transcription factor and a transcriptional activator of thesame or a different transcription factor, and (ii) a second nucleic acidencoding a reporter gene whose transcription is regulated by theDNA-binding domain of a transcription factor contained in the APP fusionprotein; and (iii) a third nucleic acid encoding a Mint 2 protein (SEQID NO:13); (b) measuring the levels of reporter gene transcription thatoccurs in the presence and absence of the test compound; and (c)comparing the levels of reporter gene transcription measured in step (b)wherein increased or decreased levels of reporter gene transcription inthe presence of the test compound indicate that the compound modulatestranscriptional activation.
 2. The method of claim 1 wherein theDNA-binding domain is from Gal4 or LexA.
 3. The method of claim 1wherein the transcriptional activation domain is from VP
 16. 4. Themethod of claim 1 wherein the reporter gene transcription levels aremeasured by measuring expression of the reporter gene.
 5. The method ofclaim 1 wherein the cell is a eukaryotic cell.
 6. The method of claim 5wherein the cell is a mammalian cell.
 7. The method of claim 6 whereinthe cell is a human cell.